The identification of human papillomavirus (HPV) as a causative agent for a host of conditions, particularly cervical cancer, has led to the development of HPV-targeting therapeutics, including therapeutic HPV vaccines, for the treatment of HPV-associated malignancies. However, the potent efficacies demonstrated by the therapeutic HPV vaccine candidates in preclinical studies are often not reflected in clinical settings. This discrepancy is potentially due to the inability of existing preclinical HPV tumor models to fully replicate the biology of clinical HPV-associated cancers. We hypothesize that an ideal preclinical HPV tumor model should possess the following characteristics: 1) forms spontaneous, localized, HPV oncogenic proteins-expressing tumors; 2) displays carcinoma morphology; 3) possesses a locally immunosuppressive tumor microenvironment (TME) resembling that of clinical HPV+ tumors; 4) tumor formation should follow clinical progression starting from a precancerous to an invasive and metastatic state; 5) be applicable to different MHC class I backgrounds; and 6) the tumor-bearing mice should respond appropriately to immunotherapeutic strategies and generate anti-tumor immunity. Preliminary data: We developed a strategy for the generation of preclinical spontaneous HPV cervicovaginal carcinoma based on orthotopic injection of oncogenic plasmids encoding HPV16-E6, HPV16-E7, constitutively active Akt, luciferase reporter gene, and Sleeping Beauty Transposase (SB) into the cervicovaginal tract of mice with electroporation to enhance transfection efficiency. Subsequent expression of SB induces the integration of plasmid DNA into the genome of transfected cells, resulting in persistent oncogenes expression and spontaneous transformation of transfected cells. In a systemic immunosuppressed setting induced by short-term anti-CD3 administration, intracervicovaginal oncogenic plasmid transfection led to the spontaneous formation of HPV+ tumors with carcinoma characteristics. We propose to further optimize our model by incorporating immunosuppressive molecules that are often overexpressed in clinical cervical cancers into our spontaneous HPV cervicovaginal tumor model and eliminate the need of short-term CD3 depletion. Also, we will further utilize genetic outbred mice and HPV16 pseudovirion delivery of oncogenes for the generation of spontaneous tumors, thereby recapitulating the genetic diverse patient population and HPV16 infection-induced oncogene introduction. Furthermore, we will examine various treatment strategies, such as the combination of therapeutic HPV vaccination with inhibitors of immunosuppressive molecules, in overcoming the immunosuppressive TME for the generation of improved therapeutic antitumor responses. Impact: A novel preclinical HPV cervicovaginal cancer model that faithfully recapitulates the clinical situation would potentiate crucial immunotherapeutic and biological research for HPV- associated cancers, provide better predictions for clinical outcomes of HPV-specific immunotherapies, and permit testing of novel molecular interventions targeting immune suppressive genes.